Effects of Dietary Steroid Saponins on Growth Performance, Serum and Liver Glucose, Lipid Metabolism and Immune Molecules of Hybrid Groupers (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu) Fed High-Lipid Diets

High-lipid diets are attributed to excessive lipid deposition and metabolic disturbances in fish. The aim of this experiment was to investigate the effects of steroidal saponins on growth performance, immune molecules and metabolism of glucose and lipids in hybrid groupers (initial weight 22.71 ± 0.12 g) fed high-lipid diets. steroidal saponins (0%, 0.1% and 0.2%) were added to the basal diet (crude lipid, 14%) to produce three experimental diets, designated S0, S0.1 and S0.2, respectively. After an 8-week feeding trial, no significant differences were found between the S0 and S0.1 groups in percent weight gain, specific growth rate, feed conversion ratio, protein efficiency ratio and protein deposition rate (p > 0.05). All those in the S0.2 group were significantly decreased (p < 0.05). Compared to the S0 group, fish in the S0.1 group had lower contents of serum triglyceride and low-density lipoprotein cholesterol and higher high-density lipoprotein cholesterol and glucose (p < 0.05). The activities of superoxide dismutase, catalase and glutathione peroxidase were significantly higher, and malondialdehyde contents were significantly lower in the S0.1 group than in the S0 group (p < 0.05). Hepatic triglyceride, total cholesterol and glycogen were significantly lower in the S0.1 group than in the S0 group (p < 0.05). Activities of lipoprotein lipase, total lipase, glucokinase and pyruvate kinase, and gene expression of lipoprotein lipase, triglyceride lipase and glucokinase, were significantly higher in the S0.1 group than in the S0 group. Interleukin-10 mRNA expression in the S0.1 group was significantly higher than that in the S0 group, while the expression of interleukin-6 and tumor necrosis factor-α genes were significantly lower than those in the S0 group. In summary, adding 0.1% steroidal saponins to a high-lipid diet not only promoted lipolysis in fish livers, but also activated glycolysis pathways, thus enhancing the utilization of the dietary energy of the groupers, as well as supporting the fish’s nonspecial immune-defense mechanism.

Aquaculture has undeniably established its crucial role in global food security and nutrition. The total aquaculture production of the world has reached 122.6 million tons in 2020 [1]. The scale of aquaculture in the world is so large that the more aquatic feeds are required. In recent years, fish-meal and soybean-meal supply have been increasingly tight, which made the prices of some miscellaneous meal fluctuate from time to time, such as cotton meal, rapeseed meal and peanut meal, etc. [2,3]. In view of such a serious situation, full utilization of the energy effect of lipids and carbohydrates in feed to save protein is a Table 1. Ingredients (g/100 g diet) and proximate composition (% dry matter).

Ingredients S 0
Fish meal 36.00 Poultry by-product meal 10.50 Soybean meal 6.00 Concentrated cottonseed protein 19.00 Wheat flour 16

Fish and Feeding Trial
The healthy hybrid groupers of consistent genetic background were purchased from a commercial hatchery (Hongxing Hatchery, Zhanjiang, China). The fish were then reared in an outdoor concrete pond (5 m × 4 m × 2 m), fed a commercial diet (50% crude protein, 8% crude lipid) and domesticated for two weeks. All fish were starved for 24 h, and then healthy groupers (initial weight 22.71 ± 0.12 g) were selected randomly and divided into 9 buckets (1 m 3 Fiberglass farming buckets) with 25 fish each. The experiment was conducted in an indoor hydrostatic water culture system at the Marine Biological Research Base of Guangdong Ocean University (Donghai Island, Zhanjiang, China). The fish, reared in three groups with three replicates, were fed the experimental diets to apparent satiation at 8:00 and 16:00 daily for 8 weeks. During the feeding trial, fish were continuously oxygenated every day, kept at a temperature of 30.5 ± 0.8 • C, a salinity of 28-32, dissolved oxygen of 5-6 mg/L and an ammonia content of <0.2 mg/L. The culture water was replaced by 80% each other day.

Sample Collection and Chemical Analysis
At the end of the feeding trial, all fish in each bucket were made to fast for 24 h. In each replicate, fish were counted and weighed to calculate survival rate (SR), percent weight gain (PGR), specific growth rate (SGR), protein efficiency ratio (PER) and feed conversion ratio (FCR). Two fish were randomly selected in each replicate for whole-fish composition analysis. Three groupers were randomly selected from each replicate and measured for body length and weight, and their dissected livers and viscera were weighed to evaluate the condition factor (CF), hepatopancreas index (HSI) and viscerosomatic index (VSI).
Six fish were randomly selected from each replicate after weighing, and blood was collected from the tail vein. The blood samples were left at 4 • C for 12 h and centrifuged at 4 • C and 3500 rpm for 15 min. The serum obtained with centrifugation was for the analysis of biochemical indexes and antioxidative enzyme activities. The soybean-sized liver was cut in each replicate and washed with saline, and then preserved in 4% formaldehyde solution for making the histological section stained with PAS. Glycogen was quantified with software IPwin32 (6.0.0.260). Four livers in each replicate were obtained, two for measuring the activity of enzymes and another two for analyzing gene expression. The analysis methods for the index are listed in Table 2.

Quantitative RT-PCR Analysis of Gene Expression
Total RNA in the liver was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA). The cDNA was synthesized by Prime Script RT kit (Takara, Osaka, Japan), and qRT-PCR was performed using SYBR Premix Ex Taq kit (Takara, Osaka, Japan) and carried out using a quantitative thermal cycler (Light Cycler480II, Roche Diagnostics, Basel, Switzerland). The reaction volume was 10 µL, containing 3.2 µL sterilized double-distilled water, 1 µL cDNA, 0.4 µL each primer and 5 µL SYBR Premix Ex Taq (Takara, Osaka, Japan). The cycle conditions were 30 s at 95 • C, then 35 cycles of 5 s at 95 • C, 25 s at 60 • C and 30 s at 72 • C. β-actin was as the reference gene to correct the results of different batches. The results were calculated using the 2 −∆∆Ct method in relative expression analysis. The primers used for qRT-PCR analysis are listed in Table 3.
Protein efficiency ratio (PER) = (final mean weight − initial mean weight)/(total feed intake × content of dietary protein).
Protein deposition rate (PDR, %) = 100 × (final mean weight × content of final body protein-initial mean weight × content of initial body protein)/(total feed intake × content of dietary protein).
All data were analyzed with a one-way analysis of variance (ANOVA) and significant differences among dietary groups were estimated by Tukey's multiple comparison test. The results were expressed as means ± standard deviation (SD). Significant differences were chosen at p < 0.05. The images of the experimental results were drawn with Graph Pad Prism 8.0 software (8.0.2.263).

Growth Performance
As indicated in Table 4, the FBW, PWG, PER, PDR and SGR of the fish in the S 0.1 group were not significantly different from the S 0 group and were significantly higher than those in S 0.2 group (p < 0.05). The number of fish that died per replicate in group S 0 was 0, 3 and 1, respectively; in group S 0.1 , it was 1, 1 and 1, respectively; and in group S 0.2 , it was 3, 3 and 2, respectively. The HSI and VSI of fish in the S 0 group were not significantly different from the S 0.2 group, but were significantly lower than those in the S 0.1 group (p < 0.05).

Whole-Body Proximate Chemical Analysis
Body composition in the initial fish in each group was similar. After 8 weeks of a feeding trial, no significant difference was found in the moisture, crude protein and crude lipid levels of the whole fish among the experimental groups (p > 0.05) ( Table 5).

Serum Biochemical Indexes
The content of TG in the S 0 group was significantly higher than that in the S 0.1 group, but significantly lower than that in the S 0.2 group (p < 0.05). The content of serum TC and LDL-C was significantly lower in the S 0.1 group than in the S 0.2 group (p < 0.05). The level of HDL-C and GLU were significantly higher in the S 0.1 and S 0.2 groups than in the S 0 group (p < 0.05) ( Table 6).

Serum Antioxidative Index
The MDA contents in the S 0 and S 0.2 groups were significantly higher than that in the S 0.1 group (p < 0.05) ( Table 7). SOD activity of the S 0 group was significantly lower than in the S 0.1 group, but significantly higher than in the S 0.2 group (p < 0.05). The CAT activity in the S 0.1 and S 0.2 groups were significantly higher than that in the S 0 group (p < 0.05). Compared to the S 0.1 group, GSH-PX activity in S 0 and S 0.2 groups was significantly decreased (p < 0.05).

Liver Histochemistry by PAS Stain
The results of the liver PAS stain are shown in Figure 1. The blue substance is the nucleus, and the purple substance is glycogen ( Figure 1A). The percentage of nucleus in all groups was not significantly different (p > 0.05). The percentage of glycogen in the control group was significantly higher than in the S 0.1 group, but was significantly lower than in the S 0.2 group (p < 0.05) ( Figure 1B).

Liver Biochemical Indexes
There was no significant difference in hepatic TP among the three groups (p > 0.05) (Figure 2). Both the TG and TC contents of the S0.1 group were significantly lower than those of the S0 group (p < 0.05). The LG of the S0 group was significantly higher than that

Liver Biochemical Indexes
There was no significant difference in hepatic TP among the three groups (p > 0.05) (Figure 2). Both the TG and TC contents of the S 0.1 group were significantly lower than those of the S 0 group (p < 0.05). The LG of the S 0 group was significantly higher than that of the S 0.1 group (p < 0.05) and was not significantly different from the S 0.2 group (p > 0.05). Quantitative data on glycogen and nucleus. Different letters on the bars indicated significant difference (p < 0.05).

Liver Biochemical Indexes
There was no significant difference in hepatic TP among the three groups (p > 0.05) (Figure 2). Both the TG and TC contents of the S0.1 group were significantly lower than those of the S0 group (p < 0.05). The LG of the S0 group was significantly higher than that of the S0.1 group (p < 0.05) and was not significantly different from the S0.2 group (p > 0.05). From Figure 3A, enzyme activities of LPL and TL in the S0.1 and S0.2 groups were significantly higher than those in the S0 group (p < 0.05). Higher HL activity was found in the S0.2 group (p < 0.05), while it was not significantly different between the S0 and S0.1 groups (p > 0.05). The activities of GK and PK were significantly higher in the S0.1 group than in From Figure 3A, enzyme activities of LPL and TL in the S 0.1 and S 0.2 groups were significantly higher than those in the S 0 group (p < 0.05). Higher HL activity was found in the S 0.2 group (p < 0.05), while it was not significantly different between the S 0 and S 0.1 groups (p > 0.05). The activities of GK and PK were significantly higher in the S 0.1 group than in the other two groups (p < 0.05). No significant difference was found in the HK activity in all groups (p > 0.05).  The lpl and atgl mRNA expressions were significantly higher in the S0.1 group than in the other two groups (p < 0.05) ( Figure 3B), while gene expression levels of lpl and atgl in the S0.2 group were significantly lower than those in the S0 group (p < 0.05). The ppar α mRNA expression did not show significant differences between the S0 and S0.1 groups (p > 0.05), while it was significantly higher in the S0.2 group (p < 0.05). The expression of glut2  The lpl and atgl mRNA expressions were significantly higher in the S 0.1 group than in the other two groups (p < 0.05) ( Figure 3B), while gene expression levels of lpl and atgl in the S 0.2 group were significantly lower than those in the S 0 group (p < 0.05). The ppar α mRNA expression did not show significant differences between the S 0 and S 0.1 groups (p > 0.05), while it was significantly higher in the S 0.2 group (p < 0.05). The expression of glut2 mRNA was significantly higher in the S 0.1 and S 0.2 groups than in the S 0 group (p < 0.05). The gk mRNA expression was significantly lower in the S 0 and S 0.2 groups than in the S 0.1 group (p < 0.05). The expression of pfk b mRNA was not significantly different between the S 0.1 and S 0 groups and was significantly lower than in the S 0.2 group (p < 0.05).

Liver Immune Molecules
As Figure 4A shows, the mRNA expressions of the genes cat and sod were significantly higher in the S 0.1 group than in the other two groups (p < 0.05). The mRNA expression of the gr was not significantly different between the S 0 and S 0.1 groups, but it was higher than in the S 0.2 group (p < 0.05). The mhc II and il-10 mRNA expressions in the S 0.1 group were significantly upregulated when compared with other groups (p < 0.05). The tgf -β mRNA expression in the S 0 and S 0.1 groups was lower than in the S 0.2 group (p < 0.05). The expressions of ifn-γ and il-6 mRNA in S 0.2 group were significantly higher than in other groups (p < 0.05). The tnf -α mRNA expression in the S 0.1 and S 0.2 groups were significantly lower than in the S 0 group (p < 0.05).

Discussion
In most fish, dietary lipids are able to promote protein deposition efficiently in vivo, which is known as the protein-saving effect. Although the appropriate dietary lipid level for hybrid groupers was 10% [17,23,24], a higher lipid level in the diet was used in the practical feed in order to realize the protein-saving effect. However, excessive dietary li-

Discussion
In most fish, dietary lipids are able to promote protein deposition efficiently in vivo, which is known as the protein-saving effect. Although the appropriate dietary lipid level for hybrid groupers was 10% [17,23,24], a higher lipid level in the diet was used in the practical feed in order to realize the protein-saving effect. However, excessive dietary lipids would cause lipid accumulation, inflammation and decreased PER and PDR in hybrid groupers, hybrid yellow catfish (Pelteobagrus fulvidraco × P. vachelli) and yellow croakers (Larimichthys crocea), inhibiting fish growth [25][26][27]. In this experiment, when 0.1% steroidal saponins was added to the diet with 14% crude lipids, more PER and PDR were obtained, but when steroidal saponins was up to 0.2%, fish growth was significantly inhibited. Studies in Atlantic salmon (Salmo salar L) [28] found that dietary addition of 0.2% soya-saponins increased fish PER and PDR, but supplementation of 0.2% or 0.32% soyasaponins decreased the weight-gain rate of carnivorous field eels (Monopterus albus) [29] and Japanese flounder [12]. Research in omnivorous carp [30] found that growth performance was significantly inhibited when dietary Momordica charantia saponins were above 0.64%. Thus, high doses of saponins were toxic to fish and decreased growth. Dietary soyasaponins over 0.25% significantly caused a decrease in the specific growth ratio and feed efficiency ratio in juvenile turbot (Scophthalmus maximus) [20]. However, these differences in the results might be related to saponin dose, species habits, feeding habits and the saponin type. The addition of 0.05-1% Panax notoginseng extract (with 80% saponins) to high-lipid diets (15%) promoted the growth of hybrid groupers. Although the growth was increased above 0.5% [31], in which the initial grouper size was larger than in the present study, we thought that the larger fish would have greater tolerance. However, the groupers of a similar size to those in the present study were fed a diet containing 0.4% saikosaponin d, which gave the best growth, and fish fed a diet containing 0.8% saikosaponin d had similar weight gain compared to the control group. Saikosaponin is a triterpenoid saponin. The saponin used in this experiment is a steroidal saponin extracted from mulberry leaves. The two types of saponin have different functions. Perhaps the type of the saponin made a difference in the dietary dose and effect on fish. The above research was combined to show that the addition of appropriate levels or type of saponins to high-lipid diets was helpful for playing a role in the conservation of protein and to increase PER and PDR, thus resulting in promoting fish growth.
Compared to terrestrial animals, aquaculture animals are characterized by a shorter digestive tract that limits the ability to digest and utilize food. High-lipid diets would increase the burden on fish and cause problems such as disorders of lipid metabolism in the serum and liver, thus affecting body health [32][33][34]. In a non-high-lipid diet, saponin reduced TC, TG and LDL-C and increase HDL-C in the serum of pacific white shrimp (Litopenaeus vannamei) [35] and juvenile turbot [36]. In the present experiment, the addition of steroidal saponins to the high-lipid diet significantly increased the enzyme activities of LPL and HL and upregulated the expression of lpl, atgl and ppar α genes in livers of hybrid groupers. Meanwhile, a decrease in TG and TC levels in serum and the liver of hybrid groupers was also observed, which is consistent with the trend of lipid-metabolizing enzymes in the liver. The saponin could activate the PPAR signaling pathway to upregulate the gene expression of lpl, atgl and ppar α to stabilize lipid metabolism disorders of hybrid groupers fed with a high-lipid diet [19]. It was indicated that steroidal saponins probably enhanced the hydrolysis of triglycerides to fatty acids through activating the PPAR signaling pathway, which subsequently activated the LDL and HDL receptor families in the cholesterol metabolic pathway to mediate cholesterol transport [18,37,38].
In addition, the intake of a high-lipid diet is often accompanied by disturbances in the glucose metabolism in fish, mainly in terms of wave blood glucose and hepatic glycogen content [33,34,39]. Saponins were found to significantly reduce hepatic glycogen and blood glucose levels and elevated the enzyme activities of glycolysis in carp that were fed with a high-starch diet [30]. In this experiment, the addition of steroidal saponins to the high-lipid diets significantly upregulated hepatic GLUT2, GK and PFK b gene expression, increased GK and PK enzyme activities, and decreased the hepatic glycogen content. When the organism was stimulated, GK, HK and PFK b expression in the AMPK signaling pathway was able to be activated, which subsequently regulated gene expression and glycolysis and provided energy to the organism [30,40]. The steroidal saponins might promote grouper growth by activating the expression of the PFK b gene in the AMPK signaling pathway and subsequently upregulating gene expression and the activity of enzymes in the glycolysis pathway. Dietary carbohydrates could be better energized by steroidal saponin stimulation, which was more conducive to the protein-sparing effect.
Saponins have been confirmed to reduce MDA content and enhance the antioxidant capacity of carp [6], white shrimp [41] and swimming crab (Portunus trituberculatus) [42], by increasing SOD, CAT, GSH-PX and GR activities and genes expression. In this study, enzyme activities and gene expression of SOD, CAT, GSH -PX and GR in groupers fed diets containing 0.1% steroidal saponins were significantly increased, and MDA levels in serum also decreased. At the same time, the expression of cell factors mhc II, il-10 and tgf-β genes were upregulated, and inf-γ, il-6 and tnf-α gene expressions were downregulated in groupers fed diets containing 0.1% steroidal saponins. In a similar trend, higher expressions of mhc II, il-10 and tgf-β genes and lower expressions of inf-γ, il-6 and tnf-α genes in the liver were found in turbot [43], sea bream [44] and carp [45,46] fed the diet with saponins. The expression of mhc II in the antigen presentation pathway might be upregulated by the saponins in groupers, which enables T cells to recognize antigens [47], together with the higher expression of il-10 and tgf-β genes to exhibit the anti-inflammation in the body's immune system. Saponins could act on the NF-κB signaling pathway to play a role in inflammation in the enterocytes [48] and also enhance the antioxidant capacity of carp [45] and mice [49] by acting on the Nrf2 signaling pathway and upregulating sod, cat and gr gene expressions, which then alleviate the liver damage. As a result, the steroidal saponins effectively improved the antioxidant ability and removed peroxide products, etc., in the body through the nonspecial immune factors maintaining the oxidative homeostasis, and thus enabled the orderly metabolism of nutrients in the body.
In conclusion, compared to the fish that were fed a diet without steroidal saponins, groupers that ingested a high-lipid diet with 0.1% steroidal saponins could achieve a better protein efficiency ratio, lower blood lipids and a healthier liver, which were all derived from raising the efficiency of the glycolipid metabolism for energy supply. Meanwhile, 0.1% steroidal saponins helped increase the nonspecial immune-defense mechanism of the fish by regulating immune molecules.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in the main article.

Conflicts of Interest:
The authors declared that they had no known competing financial interest or personal relationships that could have influenced the work reported in this paper.